Aav capsid proteins for nucleic acid transfer

ABSTRACT

Recombinant adeno-associated viral (AAV) capsid proteins are provided. Methods for generating the recombinant adeno-associated viral capsid proteins and a library from which the capsids are selected are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/829,639, filed Dec. 1, 2017, now allowed; which is a divisionalapplication of U.S. application Ser. No. 14/853,552, filed Sep. 14,2015, now U.S. Pat. No. 9,856,469; which is a divisional application ofU.S. application Ser. No. 13/594,773, filed Aug. 24, 2012, now U.S. Pat.No. 9,169,299; which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/526,688, filed Aug. 24, 2011 and to U.S.Provisional Application No. 61/545,488, filed Oct. 10, 2011, the entirecontents of each is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract HL092096awarded by the National Institutes of Health. The Government has certainrights in this invention.

REFERENCE TO SEQUENCE LISTING

The present disclosure includes a sequence listing which is beingsubmitted electronically in the form of a text file, created Apr. 22,2021 and named “091511-0642_Sequence_Listing_1.txt” (263,014 bytes), thecontents of which are incorporated herein by reference in theirentirety.

INTRODUCTION

The subject matter described herein relates to in vitro and in vivoselection of sequences from a library of sequences encoding recombinantadeno-associated viral (AAV) viral capsid proteins and to methods ofgenerating the libraries. The subject matter also relates to nucleotidesequences isolated from the libraries and to the AAV capsid proteinsencoded by these sequences, and their usefulness as capsid proteins inrecombinant AAV vectors for various nucleic acid transfer applications.The subject matter also relates to plasmids and viruses comprising theidentified sequences, which preferably provide a high transductionefficiency and a low level of neutralization by the human immune system.

BACKGROUND

Multiple recombinant gene transfer vectors based on different types ofviruses have been developed and tested in clinical trials in recentyears. Gene transfer vectors based on adeno-associated virus (AAV),i.e., AAV vectors, have become favored vectors because ofcharacteristics such as an ability to transduce different types ofdividing and non-dividing cells of different tissues and the ability toestablish stable, long-term transgene expression. While vectors based onother viruses, such as adenoviruses and retroviruses may possess certaindesirable characteristics, the use of other vectors has been associatedwith toxicity or some human diseases. These side effects have not beendetected with gene transfer vectors based on AAV (Manno et al., NatureMedicine, 12(3):342 (2006)). Additionally, the technology to produce andpurify AAV vectors without undue effort has been developed.

At least eleven AAV serotypes have been identified, cloned, sequenced,and converted into vectors, and at least 100 new AAV variants have beenisolated from non-primates, primates and humans. However, the majorityof preclinical data to date involving AAV vectors has been generatedwith vectors based on the human AAV-2 serotype, considered the AAVprototype.

There are several disadvantages to the currently used AAV-2 vectors. Forexample, a number of clinically relevant cell types and tissues are notefficiently transduced with these vectors. Also, a large percentage ofthe human population is immune to AAV-2 due to prior exposure towildtype AAV-2 virus. It has been estimated that up to 96% of humans areseropositive for AAV-2, and up to 67% of the seropositive individualscarry neutralizing anti-AAV-2 antibodies which could eliminate orgreatly reduce transduction by AAV-2 vectors. Moreover, AAV-2 has beenreported to cause a cell mediated immune response in patients when givensystemically (Manno et al., Nature Medicine, 12(3):342 (2006)).

Methods of overcoming the limitations of AAV-2 vectors have beenproposed. For example, randomly mutagenizing the nucleotide sequenceencoding the AAV-2 capsid by error-prone PCR has been proposed as amethod of generating AAV-2 mutants that are able to escape theneutralizing antibodies that affect wildtype AAV-2. However, it isexpected that it will be difficult to generate significantly improvedAAV-2 variants with single random point mutations, as the naturallyoccurring serotypes have, at most, only about 85% homology in the capsidnucleotide sequence.

Methods of using a mixture of AAV serotype constructs for AAV vectorshave also been developed. The resulting chimeric vectors possess capsidproteins from different serotypes, and ideally, have properties of thedifferent serotypes used. However, the ratio of the different capsidproteins is different from vector to vector and cannot be consistentlycontrolled or reproduced (due to lack of genetic templates), which isunacceptable for clinical use and not satisfactory for experimental use.

A third approach at modifying the AAV-2 capsid are peptide insertionlibraries, in which randomized oligonucleotides encoding up to 7 aminoacids are incorporated into a defined location within the AAV-2 capsid.The display of these peptides on the AAV-2 capsid surface can then beexploited to re-target the particles to cells or tissues that areotherwise refractory to infection with the wildtype AAV-2 virus.However, because knowledge of the atomic capsid structure is aprerequisite for this type of AAV modification, this method is generallyrestricted to AAV serotype 2. Moreover, peptide insertion librariestypically cannot address the issues of AAV particle immunogenicity ortransduction efficiency.

Thus, a need remains for new AAV vectors and a method of generating newAAV vectors. In particular, there is a need for AAV based vectors thatcan be used efficiently with a variety of cell types and tissues, andthat do not react with a pre-existing anti-AAV human immunity that couldneutralize or inactivate the vectors. Also needed are vectors thattransduce different cell types in vivo and in vitro and that offer amore restricted biodistribution or a more promiscuous biodistribution,depending on the intended use. In particular, there remains a need forvectors capable of transducing a variety of cells types, such ashematopoietic stem cells or embryonic stem cells, as well as havingdesirable properties.

A recombinant AAV vector known as “AAV-DJ” has been reported anddescribed in U.S. patent application Ser. No. 12/538,791, published asUS 20100047174, which is incorporated by reference herein, in itsentirety.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

In one aspect, recombinant capsid proteins and methods for generatingrecombinant capsid proteins are provided. The capsid proteins includeregions or domains that are derived from different serotypes of AAV. TheAAV serotypes may be human or non-human. Recombinant AAV comprising thecapsid proteins and plasmids encoding the capsid proteins are alsoprovided.

In one aspect, a capsid protein comprises a first amino acid sequencesimilar or identical to a contiguous sequence of amino acids from afirst AAV serotype, and a second amino acid sequence similar oridentical to a contiguous sequence of amino acids from at least a secondAAV serotype.

In one embodiment, the capsid protein additionally comprises a sequenceof amino acid residues similar or identical to a contiguous sequence ofamino acids from a third AAV serotype.

In another embodiment, the sequences of amino acids in the firstsequence, in the second sequence, and in the third or further sequence,are each a contiguous sequence of amino acids from the first AAVserotype, the second AAV serotype, the third and/or further AAVserotypes. In another embodiment, the contiguous sequence of amino acidsforms a conserved set of amino acid residues, the conserved set havingat least about 70% sequence identity, at least about 75% sequenceidentity, at least about 80% sequence identity, at least about 85%sequence identity, at least about 90% sequence identity, at least about95% sequence identity, at least about 96% sequence identity, at leastabout 97% sequence identity, at least about 98% sequence identity, or atleast about 99% sequence identity with the AAV serotype from acontiguous sequence in its respective AAV serotype.

In some aspects, a capsid protein is encoded by a nucleotide sequenceselected from the group of sequences consisting of SEQ ID NOs: 1-28, ora sequence having at least 95% sequence identity thereto.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 1.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 2.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 3.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 4.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 5.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 6.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 7.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 8.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 9.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 10.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 11.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 12.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 13.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 14.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 15.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 16.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 17.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 18.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 19.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 20.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 21.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 22.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 23.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 24.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 25.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 26.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 27.

In one embodiment, the capsid protein comprises an amino acid sequenceencoded by the nucleotide sequence identified by SEQ ID NO: 28.

A viral particle comprising a capsid protein sequence as describedabove, is contemplated in some embodiments. Disclosed herein is a genusof viral particles comprising the capsid proteins encoded by thenucleotide sequences identified by SEQ ID NOs: 1-28 of the sequencelisting, or a sequence having at least 95% sequence identity to saidsequences.

Also disclosed is a plasmid comprising the nucleotide sequence selectedfrom the group of sequences consisting of SEQ ID NOs: 1-28, or asequence having at least 95% sequence identity thereto.

Also disclosed is a recombinant AAV vector (rAAV), comprising a capsidprotein having an amino acid sequence encoded by a nucleotide sequenceselected from the group of sequences consisting of SEQ ID NOs: 1-28, ora sequence having at least 95% sequence identity thereto.

In one aspect, a method of expressing a gene of interest in a mammal isprovided. The present disclosure also provides a method of transfer of anucleic acid of interest into a mammal, comprising introducing arecombinant AAV vector into a mammal, the recombinant AAV vectorencoding a gene of interest which is encapsidated into a capsid proteinencoded by a nucleotide sequence selected from the group of sequencesconsisting of SEQ ID NOs: 1-28, or a sequence having at least 95%sequence identity thereto.

In still another aspect, a method of generating a library of recombinantAAV plasmids is disclosed, the method comprising: isolating AAV capsidnucleotide sequences from two or more serotypes of AAV; digesting theAAV capsid nucleotide sequences into fragments; reassembling thefragments using PCR to form PCR products; and cloning the re-assembledPCR products into plasmids to generate a library of recombinant AAVplasmids.

The present disclosure also provides a method of generating a library ofrecombinant AAV plasmids, comprising (a) isolating AAV capsid nucleotidesequences from two or more serotypes of AAV; (b) digesting the AAVcapsid nucleotide sequences into fragments; (c) reassembling thefragments using PCR to form PCR products; and (d) cloning there-assembled PCR products into a wildtype viral genome to generate alibrary of recombinant AAV vectors (rAAVs).

In another embodiment, the method comprises transfecting cells with theplasmids to produce a viral library, preferably an AAV viral library. Insome aspects, the method further comprises (e) infecting cells in vitrowith the rAAVs; (f) passaging the selected rAAVs in cells in vitro inthe presence of a stringent condition and identifying an rAAV capsidthat survives said passaging; and, optionally, (g) repeating (b) through(f) one or more times. In some aspects, the method further comprises (h)infecting a laboratory mammal in vivo with the selected rAAVs; (i)passaging the selected rAAVs in a laboratory mammal in vivo able toinfect and propagate in said laboratory mammal and identifying an rAAVcapsid that survives said passaging; and, optionally, (j) repeating (h)and (i) one or more times.

Adenovirus has a broad host range, i.e., it can infect many human andother mammalian cell lines or primary cells, including replicative aswell as non-replicative cells. Some lymphoid cell lines may be moreresistant to Adenovirus infection, and thus may need high quantities ofviruses to achieve sufficient infection levels. Cell types that may beused in the methods disclosed herein include, but are not limited to,CHO cells, monocytes, dendritic cells (DCs), freshly isolated humanblood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, Langerhanscells and dermal DCs, Human T cell leukemia DND-41 cells, p53-deficientcancer cells, tumor cells retaining wild-type p53, tumor cells ofunknown p53 status, adenocarcinomic human alveolar basal epithelialcells, also known as “A549 cells,” human KB cells, Madin Darby BovineKidney (MDBK) cells, Mouse Embryonic Fibroblasts (MEF cells), humanpulmonary artery endothelial cells (hPAEC), NIH-3T3 cells, Huh-7.5cells, Hep G2 cells, HEp-2 cells, HeLa cells, Dempsey cells, humanembryonic kidney 293 cells (also known as “HEK 293” or “293 cells”),fetal rhesus monkey kidney (FRhK-4) cells, rat hepatoma H4TG cells, LMHchicken hepatoma epithelial cells, primary human hepatocytes and primaryhuman keratinocytes. In some aspects, the rAAV is used to infect 293cells. In some aspects, the rAAV is used to infect hPAEC cells. In someaspects, the rAAV is used to infect Huh-7.5 cells. In some embodiments ahelper Adenovirus is used.

In some embodiments, humanized FRG mice are transfected with an AAV invitro selected library. In some embodiments, non-humanized FRG mice aretransfected with an AAV in vitro selected library.

In some embodiments, the method additionally includes, after thetransfecting, passaging the viral library in a selected cell type in thepresence of a stringent condition, and selecting AAV capsids thatsurvive the passaging. Passaging can be for several or multiplepassages, for example from between 2-5 or 2-10 passages.

In some embodiments, the method additionally includes, aftertransfecting an animal model, passaging the AAV library throughadditional animal models, for subsequent selection of particular AAVisolates.

In one embodiment, a stringent condition comprises the presence of humanimmune globulin.

In another aspect, a library prepared according to the methods describedabove is disclosed. In one embodiment the library is comprised ofplasmids of shuffled full-length capsid genes and in another embodimentthe library is comprised of viral particles obtained by transfecting allor a portion of the plasmid library into a selected cell, optionally incombination with an adenoviral helper plasmid. The new, selected AAVcapsid proteins described herein are useful for ex vivo or in vivo genetransfer, gene therapy, and genome editing applications, for example.

A library prepared according to these methods is also provided.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic showing a process for generating a recombinant AAVlibrary;

FIG. 2 is a flow chart summarizing a method of generating a library ofAAV capsids using AAV shuffle library selection;

FIG. 3 shows rounds 1 and 2 of AAV shuffle library selection in vitro;

FIG. 4 shows rounds 2 and 3 of AAV shuffle library selection in vitro;

FIG. 5 shows rounds 3 and 4 of AAV shuffle library selection in vitro;

FIG. 6 compares the proportion of clones in the library to theproportion in each round of selection in vitro;

FIG. 7 compares portions of several AAV serotypes and a recombinant AAVcapsid protein in the library (AAV-PAEC);

FIG. 8: compares amino acid substitutions in two rAAVs;

FIG. 9: shows various views of a predicted 3-D structure of AAV-PAEC;

FIG. 10: illustrates an experimental design of a process used to screenan AAV shuffle library in vivo;

FIG. 11: illustrates an actual experiment used to screen an AAV shufflelibrary in vivo;

FIG. 12: shows PCR amplified isolates from the AAV shuffle libraryscreen in vivo;

FIG. 13: compares the proportions of individual clones in the library tothe proportion of individual clones in the selection from rounds 1 and 4of in vivo;

FIG. 14: shows amino acid substitutions in one selected rAAV (AAV-LK01);

FIG. 15: shows various views of a predicted structure of AAV-LK01;

FIG. 16: compares residues implicated in heparin binding in AAV-2 toresidues in new rAAVs;

FIG. 17: compares several AAV serotypes to several rAAV capsid proteinsisolated using in vivo selection;

FIG. 18: shows amino acid substitutions in one selected rAAV (AAV-LK02);

FIG. 19: shows various views of a predicted structure of AAV-LK02;

FIG. 20: shows amino acid substitutions in one selected rAAV (AAV-LK03);

FIGS. 21 and 22: compare the relationship of selected rAAVs to wildtypeAAVs on the DNA level and amino acid level, respectively;

FIGS. 23A and 23B: show the Dot Blot titers of the rAAVs as compared towildtype AAVs;

FIGS. 24A and 24B: compare the transduction titers of the rAAVs towildtype AAVs in Huh7.5 cells;

FIGS. 25A, 25B and 25C: compare transduction efficiency per AAV vectorgenome (vg) of the rAAVs to wildtype AAVs in Huh7.5 cells;

FIG. 26: compares transduction efficiency of rAAVs to wildtype AAVs in293 cells;

FIG. 27: compares transduction efficiency of rAAVs to wildtype AAVs inNIH3T3 cells;

FIG. 28: compares transduction efficiency of selected rAAV isolates towildtype AAVs in MEF cells;

FIG. 29: shows the Dot Blot titers of certain rAAV isolates as comparedto wildtype AAVs;

FIG. 30: illustrates the neutralizing effects of hepatocyte growthfactor on some AAVs and rAAV isolates.

FIG. 31: shows the transduction efficiency of primary human hepatocytesby selected wildtype AAVs and rAAV isolates;

FIG. 32: compares the expression levels of recombinant human factor IX(FIX) in immunocompetent C57/BL6 mice injected with variousFIX-expressing AAVs;

FIG. 33: compares the ability of selected rAAV isolates and wildtypeAAVs to avoid neutralization by human immune globulin (IVIG);

FIG. 34: compares transduction efficiency of certain rAAV isolates towildtype AAVs in 293 cells;

FIG. 35: compares transduction efficiency of certain rAAV isolates towildtype AAVs in Huh 7.5 cells;

FIG. 36: compares transduction efficiency of certain rAAV isolates towildtype AAVs in PAEC cells;

FIG. 37: compares transduction efficiency of certain rAAV isolates towildtype AAVs in primary human keratinocytes;

FIG. 38: compares transduction efficiency of certain rAAV isolates towildtype AAVs in DND-41 cells;

FIG. 39: compares transduction efficiency of particular rAAV isolates towildtype AAVs in DND-41 cells;

FIG. 40: compares transduction efficiency of certain rAAV isolates towildtype AAVs in 3T3 cells;

FIG. 41: compares transduction efficiency of certain rAAV isolates towildtype AAVs in HeLa cells;

FIG. 42: compares transduction efficiency of certain rAAV isolates towildtype AAVs in MEF cells;

FIG. 43: compares transduction efficiency of certain rAAV isolates towildtype AAVs in H4TG cells;

FIG. 44: compares transduction efficiency of certain rAAV isolates towildtype AAVs in LMH cells;

FIG. 45: compares transduction efficiency of certain rAAV isolates towildtype AAVs in FRhK-4 cells;

FIG. 46: compares the transduction of various cell lines by several wildtype and recombinant AAV serotypes;

FIG. 47: shows relatedness of wildtype AAVs to rAAVs isolates obtainedby selection on human PAEC cells on the DNA level and amino acid level;and

FIG. 48: shows the titer and transduction efficiency of rAAVs isolatesobtained by selection on human PAEC cells as compared to wildtype AAVs.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC.

SEQ ID NO:2 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK01.

SEQ ID NO:3 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK02.

SEQ ID NO:4 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK03.

SEQ ID NO:5 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK04.

SEQ ID NO:6 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK05.

SEQ ID NO:7 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK06.

SEQ ID NO:8 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK07.

SEQ ID NO:9 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK08.

SEQ ID NO:10 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK09.

SEQ ID NO:11 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK10.

SEQ ID NO:12 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK11.

SEQ ID NO:13 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK12.

SEQ ID NO:14 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK13.

SEQ ID NO:15 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK14.

SEQ ID NO:16 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK15.

SEQ ID NO:17 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK16.

SEQ ID NO:18 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK17.

SEQ ID NO:19 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK18.

SEQ ID NO:20 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-LK19.

SEQ ID NO:21 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC2.

SEQ ID NO:22 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC4.

SEQ ID NO:23 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC6.

SEQ ID NO:24 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC7.

SEQ ID NO:25 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC8.

SEQ ID NO:26 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC11.

SEQ ID NO:27 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC12.

SEQ ID NO:28 is a nucleotide sequence encoding a novel recombinant AAVcapsid protein, referred to herein as AAV-PAEC13.

SEQ ID NO:29 is an amino acid sequence encoded by SEQ ID NO: 2(AAV-LK01).

SEQ ID NO:30 is an amino acid sequence encoded by SEQ ID NO: 3(AAV-LK02).

SEQ ID NO:31 is an amino acid sequence encoded by SEQ ID NO: 4(AAV-LK03).

SEQ ID NO:32 is an amino acid sequence encoded by SEQ ID NO: 5(AAV-LK04).

SEQ ID NO:33 is an amino acid sequence encoded by SEQ ID NO: 6(AAV-LK05).

SEQ ID NO:34 is an amino acid sequence encoded by SEQ ID NO: 7(AAV-LK06).

SEQ ID NO:35 is an amino acid sequence encoded by SEQ ID NO: 8(AAV-LK07).

SEQ ID NO:36 is an amino acid sequence encoded by SEQ ID NO: 9(AAV-LK08).

SEQ ID NO:37 is an amino acid sequence encoded by SEQ ID NO: 10(AAV-LK09).

SEQ ID NO:38 is an amino acid sequence encoded by SEQ ID NO: 11(AAV-LK10).

SEQ ID NO:39 is an amino acid sequence encoded by SEQ ID NO: 12(AAV-LK11).

SEQ ID NO:40 is an amino acid sequence encoded by SEQ ID NO: 13(AAV-LK12).

SEQ ID NO:41 is an amino acid sequence encoded by SEQ ID NO: 14(AAV-LK13).

SEQ ID NO:42 is an amino acid sequence encoded by SEQ ID NO: 15(AAV-LK14).

SEQ ID NO:43 is an amino acid sequence encoded by SEQ ID NO: 16(AAV-LK15).

SEQ ID NO:44 is an amino acid sequence encoded by SEQ ID NO: 17(AAV-LK16).

SEQ ID NO:45 is an amino acid sequence encoded by SEQ ID NO: 18(AAV-LK17).

SEQ ID NO:46 is an amino acid sequence encoded by SEQ ID NO: 19(AAV-LK18).

SEQ ID NO:47 is an amino acid sequence encoded by SEQ ID NO: 20(AAV-LK19).

SEQ ID NO:48 is an amino acid sequence encoded by SEQ ID NO: 1 (PAEC).

SEQ ID NO:49 is an amino acid sequence encoded by SEQ ID NO: 28(PAEC-13).

SEQ ID NO:50 is an amino acid sequence encoded by SEQ ID NO: 25(PAEC-8).

SEQ ID NO:51 is an amino acid sequence encoded by SEQ ID NO: 27(PAEC-12).

SEQ ID NO:52 is an amino acid sequence encoded by SEQ ID NO: 23(PAEC-6).

SEQ ID NO:53 is an amino acid sequence encoded by SEQ ID NO: 24(PAEC-7).

SEQ ID NO:54 is an amino acid sequence encoded by SEQ ID NO: 26(PAEC-11).

SEQ ID NO:55 is an amino acid sequence encoded by SEQ ID NO: 22(PAEC-4).

SEQ ID NO:56 is an amino acid sequence encoded by SEQ ID NO: 21(PAEC-2).

It is to be understood that each of the nucleotide sequences disclosedherein can be translated to predict an amino acid sequence representinga rAAV capsid protein.

DETAILED DESCRIPTION

Several embodiments of the present disclosure are described in detailhereinafter. These embodiments may take many different forms and shouldnot be construed as limited to those embodiments explicitly set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of thepresent disclosure to those skilled in the art.

I. Definitions

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes more than one protein, and reference to “a compound”includes more than one compound. In describing and claiming the presentinvention, the following terminology will be used in accordance with thedefinitions set out below.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Unlessspecifically delineated, the abbreviated nucleotides may be eitherribonucleosides or 2′-deoxyribonucleosides. The nucleosides may bespecified as being either ribonucleosides or 2′-deoxyribonucleosides onan individual basis or on an aggregate basis. When specified on anindividual basis, the one-letter abbreviation is preceded by either a“d” or an “r,” where “d” indicates the nucleoside is a2′-deoxyribonucleoside and “r” indicates the nucleoside is aribonucleoside. For example, “dA” designates 2′-deoxyriboadenosine and“rA” designates riboadenosine. When specified on an aggregate basis, theparticular nucleic acid or polynucleotide is identified as being eitheran RNA molecule or a DNA molecule. Nucleotides are abbreviated by addinga “p” to represent each phosphate, as well as whether the phosphates areattached to the 3′-position or the 5′-position of the sugar. Thus,5′-nucleotides are abbreviated as “pN” and 3′-nucleotides areabbreviated as “Np,” where “N” represents A, G, C, T or U. When nucleicacid sequences are presented as a string of one-letter abbreviations,the sequences are presented in the 5′→3′ direction in accordance withcommon convention, and the phosphates are not indicated. Thus, the termpolynucleotide sequence is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

An “isolated polynucleotide” molecule is a nucleic acid moleculeseparate and discrete from the whole organism with which the molecule isfound in nature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences in associationtherewith.

Techniques for determining nucleic acid and amino acid “sequenceidentity” also are known in the art. Typically, such techniques includedetermining the nucleotide sequence of the mRNA for a gene and/ordetermining the amino acid sequence encoded thereby, and comparing thesesequences to a second nucleotide or amino acid sequence. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100.Percent identity may also be determined, for example, by comparingsequence information using the advanced BLAST computer program,including version 2.2.9, available from the National Institutes ofHealth. The BLAST program is based on the alignment method of Karlin andAltschul. Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and asdiscussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); KarlinAnd Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); andAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, theBLAST program defines identity as the number of identical alignedsymbols (i.e., nucleotides or amino acids), divided by the total numberof symbols in the shorter of the two sequences. The program may be usedto determine percent identity over the entire length of the proteinsbeing compared. Default parameters are provided to optimize searcheswith short query sequences in, for example, blastp with the program. Theprogram also allows use of an SEG filter to mask-off segments of thequery sequences as determined by the SEG program of Wootton andFederhen, Computers and Chemistry 17:149-163 (1993). Ranges of desireddegrees of sequence identity are approximately 80% to 100% and integervalues therebetween. Typically, the percent identities between adisclosed sequence and a claimed sequence are at least 80%, at least85%, at least 90%, at least 95%, or at least 98%.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat form stable duplexes between homologous regions, followed bydigestion with single-stranded-specific nuclease(s), and sizedetermination of the digested fragments. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 80-85%, preferably 85-90%, morepreferably 90-95%, and most preferably 98-100% sequence identity to thereference sequence over a defined length of the molecules, as determinedusing the methods above. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified DNA orpolypeptide sequence. DNA sequences that are substantially homologouscan be identified in a Southern hybridization experiment under, forexample, stringent conditions, as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

In the present disclosure, a recombinant AAV vector library is provided.While wild type AAVs (on which the library is based) can replicate incells, the in vitro- and in vivo-selected isolates of the presentdisclosure are non-replicating and non-infectious. In other words, theviruses in the library contain only the Rep and Cap genes from wild typeviruses, and do not contain any other reporter sequences such as GFP.After selection of the virus of interest according to the methods setforth herein, the recombinant Cap genes from the new isolates are clonedinto plasmids for expressing the recombinant Cap proteins and packagingand production of non-replicating, non-infective vectors (a process alsoknown as “vectorizing”).

II. Chimeric AAV Capsid

Capsid proteins with regions or domains or individual amino acids thatare derived from two or more different serotypes of AAV are describedherein. A capsid protein can have a first region that is derived from orhaving high levels of sequence similarity or identity to a first AAVserotype or known recombinant AAV capsid protein (e.g., AAV-DJ), asecond region similarly derived from or having high levels of sequencesimilarity or identity to a second AAV serotype or known recombinant AAVcapsid protein, as well as third, fourth, fifth, six, seventh and eighthregions, etc. derived from or having high levels of sequence similarityor identity to another AAV serotype or known recombinant AAV capsidprotein. The AAV serotypes may be human AAV serotypes or non-human AAVserotypes, such as bovine, avian, and caprine AAV serotypes. Inparticular, non-primate mammalian AAV serotypes, such as AAV sequencesfrom rodents (e.g., mice, rats, rabbits, and hamsters) and carnivores(e.g., dogs, cats, and raccoons), may be used. By including individualamino acids or regions from multiple AAV serotypes in one capsidprotein, capsid proteins that have multiple desired properties that areseparately derived from the multiple AAV serotypes may be obtained.

In one embodiment, a capsid protein, referred to herein as “AAV-DJ”,that has an amino acid sequence comprising a first region that isderived from a first AAV serotype (AAV-2), a second region that isderived from a second AAV serotype (AAV-8), and a third region that isderived from a third AAV serotype (AAV-9), is provided. The AAV-DJcapsid protein was identified from a library of capsid proteins, using amethod described below, as well as in U.S. patent application Ser. No.12/538,791, published as US Patent Publication No. 20100047174,incorporated by reference herein, in its entirety. It will beappreciated that the AAV-DJ protein is merely exemplary of thebeneficial capsid proteins that can be obtained from a library generatedaccording to the teachings herein, where the beneficial capsid proteinspreferably have multiple desired properties that are derived frommultiple AAV serotypes.

AAV-DJ has four mismatches to the two T cell epitopes in AAV-2 whichhave recently been identified as being involved in an anti-AAV cytotoxicT lymphocyte (CTL) response in humans. Thus, recombinant AAV vectorsthat include the AAV-DJ capsid protein or a derivative thereof arelikely less immunogenic in humans than AAV-2 vectors that include theAAV-2 capsid.

Studies were conducted to confirm that infectious viral particles can beformed with AAV-DJ as the capsid. In a first study, the AAV-DJnucleotide sequence was inserted into an AAV helper plasmid that alsoexpresses the AAV-2 rep gene. 293 kidney cells were then co-transfectedwith the AAV helper plasmid and an adenoviral helper plasmid, as well asa gfp-expressing vector plasmid. For comparison, two different versionsof an AAV-2 helper were used (designated AAV-2 “old” and AAV-2 “new”)which differ in the expression levels of viral proteins. Three daysafter the co-transfection, Western blotting (with 303.9 (Rep) and B1(capsid protein)) of the 293 cell extracts revealed the presence ofpresence of Rep and capsid proteins at levels comparable to those foundin cells co-transfected with plasmids expressing the AAV-2, AAV-8, orAAV-9 capsid proteins.

In another study, particle infectivity and ability to avoidneutralization by human immune globulin (IVIG) of AAV-DJ clone wascompared to wildtypes AAV-2, AAV-8, and AAV-9. Two different versions ofan AAV-2 helper were used (designated AAV-2 old and AAV-2 new) whichdiffer in the expression levels of viral proteins. Recombinant AAVs witheither the AAV-DJ, AAV-2, AAV-8, or AAV-9 capsids were produced bytriple transfecting cells with a plasmid encoding gfp flanked by AAVinverted terminal repeats (ITRs), a plasmid encoding adenoviral helpergenes, and a plasmid encoding the AAV-2 Rep gene and either the AAV-DJ,AAV-2, AAV-8, or AAV-9 capsid protein, and then freeze-thaw lysing thecells. Each virus-containing lysate was then neutralized using a highdose (1:1 volume) of two different batches of human immune globulin(IVIG1 and IVIG2) (293 cells); (Huh-7 cells)), or three decreasinglylower doses (1:2 (high), 1:10 (med), and 1:25 (low) antiserum/virus) ofthe two different batches of human immune globulin (IVIG1 and IVIG2), ora monoclonal A20 antibody (293 cells), or a polyclonal anti-AAV-8 serum(“A8”). A20 is a monoclonal antibody that was raised against assembledAAV-2 capsids and anti-AAV-8 is a polyclonal rabbit serum raised againstassembled AAV-8 capsids. Lysates treated with PBS were used as acontrol. The virus-containing lysates were neutralized by incubating thelysates with the human immune globulin or antibody for a period of time(one hour at room temperature (20-25° C.)) and then infecting cells inthe presence of helper adenovirus. The remaining activity of the virusesafter the neutralization period was determined by titrating gfpexpression units on the cells.

In the absence of IVIG1, IVIG2, and A20, the AAV-DJ virus was at leastas infectious on 293 cells as AAV-2 and several fold more infectiousthan AAV-2 on Huh-7 cells. It was demonstrated that the AAV-DJ virus andAAV-8 were able to partially escape neutralization by IVIG, while AAV-2was not. AAV-9 had intermediate IVIG results relative to AAV-DJ/AAV-8and AAV-2, and was neutralized at high IVIG doses. AAV-2 was neutralizedby the A20 antibody, but the A20 antibody did not significantly affectAAV-DJ, AAV-8, or AAV-9. The polyclonal anti-AAV-8 antiserum neutralizedall four capsids at high or medium doses, whereas AAV-2 and AAV-DJpartially escaped neutralization at the low dose.

In summary, it was previously found that the AAV-DJ virus was moreinfectious to Huh-7 cells than the previously known most efficient AAVon Huh-7 cells (AAV-2), even in the presence of high concentrations ofhuman immune globulin. Also, the AAV-DJ virus was found to have improvedresistance to neutralization by human immune globulin relative to AAV-2.Such resistance is reasonable, given that the AAV-DJ capsid was selectedfrom a library partially based on its ability to produce virus thatresist neutralization by human immune globulin. However, the improvedresistance of the AAV-DJ virus to the A20 antibody was surprising andunexpected, because (i) it was not part of the selection schemedescribed below that was used to isolate AAV-DJ; and (ii) AAV-DJ sharessubstantial identity to AAV-2, which is neutralized by the A20 antibody.

In yet another study using human melanoma cell, in vitro infectivity ofgfp-expressing vectors from the AAV-DJ capsid gene was compared to thein vitro infectivity of eight commonly used wildtype AAVs, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, or AAV-9. The melanoma cells wereinfected with 2×10⁹ recombinant AAV particles of each serotype and gfpexpression was visualized three days later. The results werequantitated, expressed as gfp expression in IU/mL, from virus titrationon the melanoma cells (in 96-well plates) and the AAV-DJ vector wassuperior to the wildtype vectors, and, notably, substantially betterthan AAV-2.

A number of cell lines were infected with ten-fold serial dilutions ofeach serotype, or AAV-DJ or the DJ heparin mutant DJ/8, discussed below,expressing a gfp reporter gene. Vector preparations were normalized tocontain 2×10⁹ total (vector DNA-containing) particles per mL prior toinfection. Three days later, gfp-expressing cells were counted andinfectious titers determined, taking into account the dilution factor.AAV-DJ vectors showed the highest infectivity on many cell lines, andratios of total to infectious particles were frequently far below 500,highlighting the extreme efficiency of AAV-DJ in vitro, and suggestingits particular usefulness for ex vivo gene transfer applications.

Vectors prepared with the AAV-DJ capsid were also tested in vivo forexpression of a gene of interest. In a first study, recombinant humanfactor IX (FIX)-expressing AAVs with either the AAV-DJ, AAV-2, AAV-8, orAAV-9 capsids were produced by a triple transfection technique. Doses of5×10¹⁰, 2×10¹¹, and 1×10¹² (low, medium, and high, respectively)recombinant viral particles were injected peripherally intoimmunocompetent mice (C57/BL6) and plasma hFIX was monitored for up tofour months after injection. The FIX protein plasma levels werequantified by ELISA.

FIX levels over 1% are considered therapeutic in hemophilics. TheAAV-8,-9 or -DJ vectors exceeded the 100% level already at the lowestdose. A dose-dependent expression from the AAV-DJ capsid at levelsequivalent to AAV-8 and -9, the two naturally identified AAVs reportedin liver, was observed. The three viruses readily outperformed the AAV-2prototype at any dose and expressed over 100% of normal hFIX levels fromintravenous injection of 5×10¹⁰ particles, whereas AAV-2 expression wasover 100% of normal hFIX levels only at a dose of 1×10¹².

In another study, recombinant human alpha-1-antitrypsin(hAAT)-expressing AAVs were prepared, from the AAV-DJ, AAV-2, AAV-8, orAAV-9 capsids. The hAAT gene was under an RSV promoter. Mice (C57/BL6)were injected via tail vein infusions of 2×10¹¹ particles and plasmalevels of hAAT were determined via specific ELISA 3, 7, and 14 daysafter injection. AAV-8, AAV-9, and AAV-DJ expressed efficiently andequally outperformed the vector with an AAV-2 capsid.

In another in vivo study, liver transduction in the presence of humanserum was quantified, to assess the ability of AAV-DJ to evadeneutralization in vivo. Mice were passively immunized with 4 or 20 mgIVIG prior to infusion of hFIX-expressing AAV-2,-8,-9, or -DJ. PlasmahFIX levels for each AAV serotype were expressed as percentcorresponding virus level in control mice treated withphosphate-buffered saline rather than IVIG as a function of time postinfusion. AAV-2 expression was completely abolished, howevertransduction with AAV-DJ,-8 or -9 was inhibited in a dose-dependentmanner, with AAV-DJ showing intermediate resistance at the high, andefficient evasion (similar to AAV-8 and AAV-9) at the low IVIG dose.These results were confirmed with a second independent IVIG batch fromanother vendor (Carimune 12%, Behring AG, data not shown).

In another study, the feasibility to repeatedly administer the differentviruses to mice was assessed, to evaluate capsid cross-neutralization.No gene expression upon re-infusion of any of the capsids into animalsalready treated with the same serotype was observed. However, AAV-8 and-9 also efficiently blocked each other, substantiating previous data(Gao, G. et al., J. Virol., 78:6381-6388 (2004)). This result mightargue against the use of vectors based on these wildtypes inre-administration protocols, albeit they could be combined with AAV-2.In contrast, primary infusion of AAV-DJ allowed subsequent expression(up to 18%) from AAV-2,-8 or -9, likely due to the fact that AAV-DJ onlyshares a limited number of epitopes with each wildtype virus. In thereverse experiment, AAV-DJ vectors were inhibited in animals immunizedwith AAV-8 or -9, while giving detectable expression in AAV-2-treatedmice. This implied a stronger or broader immune response from primaryinfusion of serotypes 8 or 9. AAV-DJ was more resistant to thecorresponding mouse sera in culture. Less cross-reactivity between AAV-8and -9 was noted.

AAV-DJ, as well as other recombinant protein capsids identified in thelibrary discussed below, retained a heparin binding domain (HBD) fromthe AAV-2 parent. This domain functions in binding to the primary AAV-2receptor heparin sulfate proteoglycan (Summerford, C. et al., J. Virol.,72:1438-1445 (1998)). To investigate the role of the AAV-DJ HBD, twocrucial arginine residues (Kern, A. et al., J. Virol., 77:11072-11081(2003)) were mutated to the respective residues in AAV-8 or -9, and arereferred to herein as AAV-DJ/8 and AAV-DJ/9. gfp expression was reducedby several orders of magnitude, and was as low as that observed withserotypes AAV-8 or AAV-9.

The infectivity drop was shown to correlate with a reduced binding tocells. A titration of infectious particles on 293 kidney cellsillustrated the role of the HBD for infection in culture, as seen by thereduction in infectivity in the HBD mutants AAV-DJ/8 and AAV-DJ/9.Additional mutants were prepared and tested, and are identified hereinas AAV-2/8 (HBD negative), AAV-8/2 (HBD positive), and AAV-9/2 (HBDpositive). Cell binding assays confirmed the role of the HBD forattachment to cultured cells, where the drop in binding with the mutantscorrelated with the transduction data. The HBD-positive AAV-8 and AAV-9mutants bound several fold more efficiently than AAV-2 on HeLa cells buttransduced less efficiently. Thus, cell attachment alone cannot explainthe unusual infectivity of AAV-DJ. Instead, a synergistic effect fromsharing beneficial properties from AAV-DJ parents is contemplated,resulting in enhancement of multiple steps in AAV-DJ transduction. TheHBD also was shown to influence biodistribution, as shown in Table 1.

TABLE 1 Relative transduction of non-hepatic tissues with AAV vectorsLung Heart Kidney Spleen Brain Pancreas Gut Muscle AAV-2 1e12 nd 0.7 ±0.1 0.8 ± 0.1 0.2 ± 0.0 nd nd nd nd 7e12 nd 1.5 ± .03 2.0 ± 0.3 1.0 ±0.2 nd nd nd nd AAV-8 1e12 0.5 ± 0.0 1.2 ± 0.2 0.9 ± 0.2 0.3 ± 0.0 0.2 ±0.0 0.2 ± 0.0 0.3 ± 0.0 0.7 ± 0.1 7e12 2.5 ± 0.3 2.5 ± 0.2 2.6 ± 0.3 1.5± 0.2 1.5 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 1.9 ± 0.2 AAV-9 1e12 0.7 ± 0.1 1.3 ±0.2 1.1 ± 0.2 0.4 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.8 ± 0.1 7e12 2.6± 0.3 3.6 ± 0.4 3.8 ± 0.4 1.5 ± 0.2 1.8 ± 0.2 1.3 ± 0.2 1.9 ± 0.2 3.0 ±0.3 AAV-DJ 1e12 0.2 ± 0.0 1.3 ± 0.2 0.8 ± 0.2 0.5 ± 0.1 nd 0.1 ± 0.0 0.1± 0.0 0.2 ± 0.0 7e12 0.6 ± 0.1 2.3 ± 0.2 2.1 ± 0.2 1.5 ± 0.2 0.4 ± 0.00.5 ± 0.0 0.5 ± 0.0 0.8 ± 0.1 AAV-DJ/8 1e12 0.6 ± 0.0 1.3 ± 0.2 0.8 ±0.2 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.7 ± 0.1 7e12 2.6 ± 0.3 2.5± 0.3 2.3 ± 0.3 1.6 ± 0.3 1.8 ± 0.2 1.2 ± 0.2 1.3 ± 0.2 2.0 ± 0.2 Vectorcopy numbers (per diploid genomic equivalent) were determined viaPhosphoimager scan analyses of Southern Blots. At least threeindependent mice were analysed per dose. Copy numbers are shown inpercent (rounded to one decimal, plus standard deviations) relative tothose in liver within each group, allowing comparison between vectorsand doses. For AAV-2, most signals were below the detection limit of theSouthern Blot analyses (~0.03 copies of double-stranded AAV DNA percell), preventing calculation of relative transduction in these cases(nd = not determined). Bolded text highlights doses/tissues whererelative AAV-DJ transduction differed by at least 2-fold from serotypes8 and 9, as well as the AAV-DJ HBD mutant.

AAV-8 and -9 (HBD-negative) demonstrated an unrestricted tropism,readily transducing all tested tissues at 1×10¹² particles per mouse. Instriking contrast, AAV-2 and likewise AAV-DJ (both HBD-positive) wererestricted to liver and, to a lesser extent, heart, kidney and spleen,and near or below detection limit in other tissues. Quantification ofdouble-stranded vector DNA (using liver as an internal standard in eachgroup) showed that AAV-DJ transduced lung, brain, pancreas and gut about2- to 4-fold less efficiently than wildtypes 8 or 9. The effect of theHBD on viral tropism was best exemplified by comparing AAV-DJ to theDJ/8 mutant: HBD deletion alleviated the liver restriction and expandedtransduction to nonhepatic tissues, identical to AAV-8 and -9, andincluding the brain. These findings corroborate and explain a series ofreports on wide tissue dissemination of vectors based on HBD-negativenatural serotypes (AAV-1 and -4 to -9) in mice, dogs and monkeys, incontrast to the HBD-positive AAV-2. Notably, AAV-DJ also transducednonhepatic tissues at the maximum dose of 7×10¹² particles, but still toa lesser extent than the HBD-negative viruses, in particular AAV-9. Evenat this dose, brain and also lung transduction remained marginal.

While the embodiments described above are primarily with respect to anrAAV capsid protein, it is recognized that capsids having amino acidand/or nucleotide sequences that are similar in sequence and having thesame function may be used and are contemplated. In one embodiment, arecombinant capsid protein having at least about 60% sequence identity,further at least about 70% sequence identity, at least about 75%sequence identity, at least about 80% sequence identity, at least about85% sequence identity, at least about 90% sequence identity, at leastabout 95% identity, at least about 96% sequence identity, at least about97% sequence identity, at least about 98% sequence identity, or at leastabout 99% sequence identity to the amino acid sequences identified inthe sequence listing is contemplated.

It will be appreciated that conservative amino acid substitutions may bein the polypeptide sequence, to achieve proteins having, for example,60%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identityto a polypeptide encoded by a nucleotide sequence disclosed herein, andpreferably with retention of activity of the native sequence.Conservative amino acid substitutions, as known in the art and asreferred to herein, involve substituting amino acids in a protein withamino acids having similar side chains in terms of, for example,structure, size and/or chemical properties. For example, the amino acidswithin each of the following groups may be interchanged with other aminoacids in the same group: amino acids having aliphatic side chains,including glycine, alanine, valine, leucine and isoleucine; amino acidshaving non-aromatic, hydroxyl-containing side chains, such as serine andthreonine; amino acids having acidic side chains, such as aspartic acidand glutamic acid; amino acids having amide side chains, includingglutamine and asparagine; basic amino acids, including lysine, arginineand histidine; amino acids having aromatic ring side chains, includingphenylalanine, tyrosine and tryptophan; and amino acids havingsulfur-containing side chains, including cysteine and methionine.Additionally, amino acids having acidic side chains, such as asparticacid and glutamic acid, are considered interchangeable herein with aminoacids having amide side chains, such as asparagine and glutamine.

A mouse model system that is severely immunodeficient has beendeveloped. These fumarylacetoacetate hydrolase (Fah)-deficient mice canbe pretreated with a urokinase-expressing adenovirus, and then highlyengrafted (up to 90%) with human hepatocytes from multiple sources,including liver biopsies. Furthermore, human cells can be seriallytransplanted from primary donors and repopulate the liver for at leastfour sequential rounds. The expanded cells displayed typical human drugmetabolism. This system provides a robust platform to producehigh-quality human hepatocytes for tissue culture. It may also be usefulfor testing the toxicity of drug metabolites and for evaluatingpathogens dependent on human liver cells for replication. (Azuma, etal., (2007) Nature Biotech. 25:903-910).

A humanized mouse model, known as FRG mice (Yecuris Corporation,Portland, Oreg.), has been designed to allow researchers to grow andexpand populations of human hepatocytes in vivo for research and drugtesting. The FRG model has the genes Fah, Rag, and Ilrg knocked out.Knocking out Fah yields mouse liver damage, the lack of Rag removes thepart of the innate immune system that rejects other mouse cells, andknocking out Ilrg inactivates the part of the immune system that wouldprevent engraftment of cells from other species including humans. Thus,the FRG mouse can either be repopulated with human donor cells of choiceor repopulated from a pool of prequalified donors. Animals can beprovided with human hepatocytes that range from 5-95% of the total livermass. Nonrepopulated FRG mice are also available for use as studycontrols.

In one embodiment, the recombinant AAV capsid protein is comprised of afirst sequence of amino acid residues from a first AAV serotype, and atleast a second sequence of amino acid residues from a second AAVserotype. The first sequence is, in the embodiment, a conserved set ofamino acids from a contiguous sequence of amino acids from the first AAVserotype. The second sequence is a conserved set of amino acids from acontiguous sequence of amino acids from the second AAV serotype. A“conserved set” of amino acids refers to a contiguous sequence of aminoacids that is identical or closely homologous to a sequence of aminoacids in the AAV serotype. In one embodiment, close homology intends atleast about 80% sequence identity. In one embodiment, close homologyintends at least about 90% sequence identity. A contiguous sequence ofamino acids in such a conserved set may be anywhere from 2 to 500, 2 to400, 2 to 300, 2 to 200, 2 to 100, or 2 to 50 amino acid residues inlength.

It will also be appreciated that the recombinant vectors describedherein are contemplated for use in methods of expressing a gene ofinterest in a variety of cells and in a mammal. Transduction into cellslines in addition to the cell lines described herein are exemplary, andother cells lines, particularly stem cells, are contemplated. In termsof in vivo use, the method preferably comprises introducing arecombinant AAV (rAAV) into a mammal, the recombinant AAV vectorencoding the gene of interest and comprising a capsid protein having anamino acid sequence selected from the group of sequences identified inthe sequence listing accompanying the present disclosure. The vectorexpressing a gene of interest is introduced to the mammal, typically byinjection, intravenously, subcutaneously, parenterally, or the like. Thegene of interest can be any gene, and many suitable genes for expressionfor therapeutic or non-therapeutic purposes are readily identified by askilled artisan. The nucleotide sequence of the gene of interest istypically “operably linked” to one or more other nucleotide sequences,including but not limited to the gene for a selected capsid protein, apromoter, and enhancer, and the like.

A gene is “operably linked” to another nucleotide sequence when it isplaced in a functional relationship with another nucleotide sequence.For example, if a coding sequence is operably linked to a promotersequence, this generally means that the promoter may promotetranscription of the coding sequence. Operably linked means that the DNAsequences being linked are typically contiguous and, where necessary tojoin two protein coding regions, contiguous and in reading frame.However, since enhancers may function when separated from the promoterby several kilobases and intronic sequences may be of variable length,some nucleotide sequences may be operably linked but not contiguous.Additionally, as defined herein, a nucleotide sequence is intended torefer to a natural or synthetic linear and sequential array ofnucleotides and/or nucleosides, and derivatives thereof. The terms“encoding” and “coding” refer to the process by which a nucleotidesequence, through the mechanisms of transcription and translation,provides the information to a cell from which a series of amino acidscan be assembled into a specific amino acid sequence to produce apolypeptide.

III. Generation of a Library of Novel AAV Capsids

In another aspect, a method of generating a library of novel AAV capsidsis provided. Embodiments of this aspect include a method of isolating arecombinant AAV plasmid that includes a novel AAV capsid.

A method of generating a library of novel rAAV capsids is provided bythe present disclosure, including the figures and sequence listing.Isolated nucleic acids encoding capsid genes are obtained using primersdesigned to include a serotype-specific part fused with common signatureregions that flank the capsid nucleic acid sequence. Then, the isolatednucleic acids are digested or fragmented, such as with DNAseI, intofragments of, for example, between about 0.2 and about 1.0 kb. Thefragments are then re-assembled into larger pieces by performing PCR,such as with Taq polymerase, in the absence of additional primers.Because of the related nature of the fragmented genes, the genefragments have overlapping regions of homology that allow the fragmentsto self prime in the absence of additional primer. After multiple roundsof PCR, products having a length approximately equal to that of theoriginally capsid genes are obtained. The PCR products include hybridproducts that contain novel rAAV capsid regions.

The full length PCR products are then PCR amplified, with Platinum Pfxpolymerase or other polymerase, using primers that bind to the signatureregions that are contained in the full length PCR products because theywere present in the original primers used to isolate the capsid nucleicacid sequences. The PCR products from this amplification step are thencloned into a conventional plasmid, to provide a library of novel AAVcapsid genes. In one embodiment, the capsid genes are cloned into anITR-rep-containing AAV plasmid, to subsequently create the actual virallibrary.

A method of isolating a recombinant AAV that includes a novelrecombinant AAV capsid, i.e., a “rAAV capsid” is isolated as describedabove. Hybrid capsid sequences are cloned into a plasmid that is capableof producing an infectious AAV genome, such as a plasmid comprising theAAV-2 rep gene, as well as the two AAV-2 ITRs. The plasmid library istransfected into cells, in some embodiments with an adenoviral helperplasmid to produce virus. The virus is then amplified in cells in thepresence of a helpervirus, such as wildtype Adenovirus-5 helpervirus.The virus may be amplified in the presence of one or more forms ofselective pressure, such as in the presence of human immune globulin.The viruses that survive multiple passages under the selective pressureare chosen for further study or use.

This approach was used to generate a library for selection in vitro onhPAEC cells. In brief, the capsid gene from ten different AAV serotypes(AAV-1, AAV-2, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, avian AAV, andbovine AAV) was fragmented, and the PCR products were blunt cloned intothe pCR4-TOPO plasmid, available from Invitrogen. Twenty-four (24)subclones were sequenced to confirm that capsid sequences that are ahybrid of different serotypes were created. Sequences from all ten ofthe serotypes were represented in the subclones. Typically, the hybridcapsid sequences included sequences from at least two, and often, morethan six, of the serotypes. The capsid sequences in the pCR4-TOPOplasmid were then subcloned into a plasmid comprising the AAV-2 repgene, as well as the two AAV-2 ITRs, that was then used to transformbacteria. It is estimated that approximately a library of 3×10⁴ hybridAAV capsid gene variants were obtained from a single reaction and from10 plates of bacteria. Up-scaling (including plating on 100 plates ofbacteria) resulted in a plasmid library of approximately 6.9×10⁵ clones.This plasmid library was then co-transfected into 293 human embryonickidney cells together with an adenoviral helper plasmid, to produce aviral library of hybrid AAV particles.

Various amounts of purified shuffled AAV were incubated with PAEC cellsat a range of MOIs (Multiplicity Of Infection), and subsequentlyinfected with helper virus needed for AAV replication (in this case wildtype human Adenovirus). Ideally, the Adenovirus would lyse the cellswithin three days, giving the AAV sufficient time to replicate and newlysynthesized Ad virus was released into the media. Media was collected 3days after infection and used directly for western blot analysis usingantibody specific for AAV CAP proteins VP1, VP2, and VP3. In order toavoid cross-packaging (a phenomenon specific to closely related AAVs),the sample with the lowest, but detectable, level of VP proteins basedon the Western blot was selected for the next round of selection. Thishelped to optimize the stringency of the library in each amplificationround, by ensuring that a single viral genome was delivered to eachcell, and subsequently packaged into the capsid expressed from its owngenome. Before the next round of selection, the supernatant was heatedto 65 C for 30 min., to inactivate more temperature-sensitive Ad viruswithout affecting the AAVs present in the sample.

The selected library of AAV capsid variants was then co-infected withwildtype Adenovirus-5 helpervirus and successfully amplified in on ofseveral possible cell lines known in the art. Successful amplificationof the viral library was confirmed by Western blots of whole cellextracts using the B1 antibody which recognizes an eight amino acidepitope that is largely conserved over most known AAV serotypes, andthus should be present in the majority of the hybrid AAVs describedherein. Replicating AAV particles were detected in all of the testedcell lines for up to five consecutive passages. Whole freeze-thaw cellextracts were used for infecting fresh cells each time. To date, theviral library has also been successfully passaged six times in primaryhuman hepatocytes, which are notoriously difficult to infect withvectors based on wildtype AAVs.

The viral library was also amplified in human Huh-7 cells in thepresence of human immune globulin (IVIG). It was found that the specificIVIG used (IVIG Gamimune® N 10% from Bayer) contained abundantneutralizing antibodies against AAV-2 and AAV-3, as well as someantibodies against AAV-1, AAV-4, AAV-5, and AAV-6. Thus, amplificationin human Huh-7 cells in the presence of IVIG provided a selectivepressure for AAV hybrids comprising domains from different serotypessince selecting for a high efficiency infection of Huh-7 cells favorsAAV-2 domains, while selecting for escape from IVIG neutralizationfavors AAV-8 and AAV-9 domains. The selection was successful, as it wasfound that with increasing passages of the library, an increasingtolerance to IVIG was achieved. After the fourth passage, survivingvirus could be amplified in the presence of 500 μL IVIG, while after thefirst passage, surviving virus could only be amplified in the presenceof approximately 10 L IVIG.

After the 5^(th) passage, the hybrid capsid sequences were PCR amplifiedand blunt cloned in pCR4-TOPO. The capsid sequences from 96 colonieswere sequenced and found to be identical. The hybrid capsid sequence isthe AAV-DJ sequence described above. Thus, a plasmid library was createdusing DNA Family Shuffling (Crameri, et al., Nature, 391: 288-291(1998)) of parental AAV capsid genes. Subsequently, a viral library wasgenerated, by transfecting the plasmid library into cells together withan adenoviral helper plasmid. This second viral library was thensubjected to selection pressure, to isolate specific candidates. Fromthose, selected shuffled capsid genes were isolated and subcloned intoan AAV helper plasmid, to make recombinant AAV vectors comprising thehybrid capsid. More particularly, DNA Family shuffling was used tocreate a complex library of hybrid particles from ten differentwildtypes. Serial amplification on human cells enriched hybrids from amultitude of AAV serotypes. The AAV-2-8-9 chimera referred to as AAV-DJwas found to be superior to natural AAVs in cultured cells andoutperformed the AAV-2 prototype in tissue in vivo. Vectors with anAAV-DJ capsid were superior in vitro and gave a robust and specific invivo performance, and provided an ability to evade humoralneutralization by human serum. Furthermore, several isolates from the invitro- and in vivo-selected AAV libraries generated according to themethods described herein were found to outperform the AAV-DJ capsidpreviously described.

After several rounds of selection, a single clone was observed. This newAAV isolate was dubbed “AAV-PAEC.” Later, additional AAV-PAECs wereisolated, and AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8,AAV-PAEC11, AAV-PAEC12 and AAV-PAEC13 isolates are identified herein.

The AAV library was also screened in vivo in humanized FRG mice. Animalswere injected with different amounts of AAV library, followed byinjection of a fixed amount of wtAd5 virus. After three days, animalswere sacrificed, and their livers extracted, homogenized and frozen inaliquots. One aliquot from each animal was used for analysis by westernblot using anti-VP1-2-3 CAP antibody for detection. Once the animal withthe lowest, but detectable signal, was identified, another frozenaliquot of liver from that animal was processed, and cleared liverlysate was injected in different amounts into another group of animals.As in the in vitro experiment described in Example 1, in order toinactivate hAd5 present in the lysate, the liver lysate was incubated at65 C for 30 min. prior to injection into another cohort of animals.

With each round of selection, 100-150 clones were sequenced. AAV DNA wasisolated from liver lysate and used to sequence the CAP genes present inthe pool. The library was found to be highly variable in the earlystages of in vivo selection, whereas in the later rounds of selection, apositive selection for certain AAV clones in the AAV library clearlyoccurred.

Several novel rAAVs with high efficiency were identified and sequenced.Also valuable are specific novel rAAV serotypes selected in vitro inhuman pulmonary arterial endothelial cells.

IV. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting. For technical procedures, reference can be madeto U.S. patent application Ser. No. 12/538,791, published as US20100047174, which is incorporated by reference herein, in its entirety,as well as to Grimm, D. et al., (Blood, 102:2412-2419 (2003)).

Example 1 AAV Capsid Library Generation

A. Plasmids for AAV Capsid Library Generation

Plasmids containing full-length capsid (cap) genes of ten different wildtype (wt) AAV serotypes were obtained, namely,AAV-1,-2,-3B,-4,-5,-6,-8,-9, avian and bovine AAV and goat AAV, whichwas partly synthesized (GeneArt, Regensburg, Germany) as a 888 ntfragment (nt 1023 to 1910). This subclone spans the entire right half ofthe goat AAV capsid protein, which comprises all 42 reported differencesbetween goat AAV and AAV-5. These cap genes were initially amplified viaPCR and subcloned into pBlueScript II SK (Stratagene). (See FIG. 1). Thepurpose was to flank all cap genes with sites for the unique restrictionenzymes Pac I (5′) or Asc I (3′), to facilitate later cloning of“shuffled” cap genes into a wildtype AAV plasmid. All primers alsocontained either a Hind III (5′) or a Spe I (3′) site, to allow directedcloning into pBlueScript (none of the four restriction enzymes cuts inany parental cap gene). A 20 nt signature region was inserted betweenthe two restriction sites in each primer, to provide conserved primerbinding sites for later PCR amplification of shuffled genes.

In parallel, a wildtype cap recipient plasmid was engineered to containthe AAV-2 packaging elements (ITRs) flanking the AAV-2 rep gene(encoding AAV replication proteins), together with Pac I and Asc I sitesfor cap cloning, and the AAV-2 polyadenylation site. Therefore, AAV-2rep (nt 191 to 2189) was PCR amplified using primers containing Bgl IIsites and then subcloned into pTRUF3 (carrying AAV-2 ITRs with adjacentBgl II sites).

B. DNA Family Shuffling of AAV Capsid Genes

For DNA shuffling of AAV capsid genes, a 2-step protocol was used wherethe parental genes were first fragmented using DNase I enzyme and thenreassembled into new full-length genes via primer-less PCR. This wasfollowed by a second PCR including primers binding outside of the capgenes, allowing their subcloning into the wildtype recipient ITR/repplasmid for packaging into an AAV library. Initially, all cap genes wereisolated from the subclones via Hind III/Spe I digestion (Eco RI forgoat AAV) and then reaction conditions were optimized as follows.Various DNAse I concentrations and incubation times were tested, aimingto obtain a pool of fragments between 0.2 and 1.0 kb in size. Optimalconditions found were: 1 μg per cap gene, 1 μL 1:200 pre-diluted DNase I(10 II/μL, Roche), 50 mM Tris Cl pH 7.4, 1 mM MgCl₂, total volume of 50μL. The reaction was incubated for 2 min at room temperature and thenstopped by heat inactivating at 80° C. for 10 min. Fragments of thedesired sizes were isolated by running the entire reaction on a 1%agarose gel (total final volume ˜60 μl). The re-assembly PCR reactionwas then optimized by testing various DNA polymerases (Pfx Platinum,Stratagene; DeepVent, NEB; Taq, Amersham) and respective conditions.Best results were obtained using PuReTaq Ready-To-Go PCR Beads(Amersham) and the following conditions: 25 μL purified cap fragments,program: 4 min 95° C., 40 cycles (1 min 95° C., 1 min 50° C., 3 min 72°C.), 10 min 72° C., 10 min 4° C. Agarose gel (1%) analysis of 1 μL fromthis reaction typically showed a smear up to 5 kb and no distinct bands.The same three polymerases as above were then evaluated for theprimer-containing second PCR, and the following conditions were foundoptimal: 1 μL Pfx Platinum, 2 μL product from first PCR, 1 mM MgSO4, 1μg of each primer (see below), 0.3 mM each dNTP, total volume 50 μL,program: 5 min 94° C., 40 cycles (30 sec 94° C., 1 min 55° C., 3 minutes68° C.), 10 min 68° C., 10 min 4° C. The primers used bound to the 20 ntsignature regions described in the previous chapter. This reaction gavea distinct ˜2.2 kb full-length cap band (1% agarose gel), which waspurified (60 μL total) and cloned (4 μL) using the Zero Blunt TOPO PCRcloning kit (with electro-competent TOP10 cells) (Invitrogen, Carlsbad,Calif., USA). This intermediate cloning step significantly enhanced theyield of shuffled cap genes, as compared to efforts to directly clonethe PCR product via conventional means (data not shown). The shuffledcap genes were then released from the TOPO plasmid via Pac I and Asc Idouble digestion and cloned into the appropriately digested ITR/reprecipient plasmid. Performing these reactions under minimal conditions(volumes and amounts), a library of approximately 3×10⁴ bacterialcolonies was obtained. Up-scaling of each step (including final platingon 100×15 cm plates) resulted in a final library of ˜6.9×10⁵ plasmidclones. Its integrity, genetic diversity and functionality was confirmedby DNA sequencing and small scale expression studies. From the latter,it was determined by extrapolation that the viral library (below)retained >90% viability.

C. Selective In Vitro Amplification of the Capsid Library

Experimental Design:

Various amounts of purified shuffled AAV were incubated with differentcell lines (in 6 cm dishes), together with varying amounts of helperAdenovirus type 5. Ideally, the Adenovirus would lyse the cells withinthree days, giving the AAV sufficient time to replicate. The AAV amountswere adjusted to obtain minimal signals in Western blot analyses of cellextracts. This helped to optimize the stringency of the library in eachamplification round, by ensuring that a single viral genome wasdelivered to each cell, and subsequently packaged into the capsidexpressed from its own genome.

In the present study, cells of interest (PAEC cells) were infected withAAV library at range of MOIs (Multiplicity Of Infection) andsubsequently infected with helper virus needed for AAV replication (inthis case wild type human Adenovirus). AAV replication depends on Advirus replication. Ad virus leads to cell lysis and release of newlysynthesized Ad virus into the media. This process releases also newlysynthesized AAV viruses. Media was collected 3 days after infection andused directly for western blot analysis using antibody specific for AAVCAP proteins VP1, VP2, and VP3. In order to avoid cross-packaging (aphenomenon specific to closely related AAVs), the sample with thelowest, but detectable, level of VP proteins based on the Western blotwas selected for the next round of selection. Before the next round ofselection, the supernatant was heated to 65 C for 30 min., to inactivatemore temperature-sensitive Ad virus without affecting the AAVs presentin the sample.

D. AAV Protein Analyses

Western blot and immunofluorescence analyses were carried out asreported (Grimm, D. et al., Blood, 102:2412-2419 (2003)) using themonoclonal B1 antibody for detection of immobilized AAV capsid proteins,useful because its eight amino acid epitope is largely conserved acrossknown AAV serotypes.

Western blots from Round-1 and Round-2 of AAV library selection on humanPAEC cells are shown in FIG. 3. Two AAV libraries, called “Library 1” or“Lib1” and “Library 2/3” or “Lib2/3” were used in the screen. Library 1had significantly lower titer than Library 2/3. In FIG. 3, the numbersabove each lane indicate the amount in microliters [μl] of each libraryused per 500 μl total infection, and “+Ad” and “−Ad” indicate (−)Library control groups treated (+) or not treated (−) with Ad,respectively. Samples selected for next rounds of AAV library selectionare indicated by boxes, as well as bold and underlined type.

Similarly, FIG. 4 shows Western blots from Round-2 and Round-3 of AAVlibrary selection. The specific signal in samples from Library 2/3 canbe seen to be weak, while specific signal in the samples from Library 1are strong. FIG. 5 shows data from Round-3 and final Round-4 of the AAVlibrary selection in hPAEC cells.

At each round of selection, the supernatant harvested was used toisolate AAV DNA for sequencing analysis of the CAP genes present in thepool. Twenty-five to thirty random clones were sequenced at each round,including the starting Library1. Results are shown in FIG. 6, whichindicates the percentage of the pool each different clone represents, ateach round of selection. As can be seen in FIG. 6, all twenty-fiveclones sequenced from Library 1 were different, while with each round ofselection specific accumulation of a single clone can be observed. AfterRound-4, all thirty clones sequenced were of identical sequence. Thisnew AAV isolate was dubbed “AAV-PAEC.”

FIGS. 7 and 8 show the relatedness of the AAV-PAEC to wild-type AAVserotypes. FIG. 7 illustrates that AAV-PAEC is most closely related toAAV1 and AAV6, with the most 5′ region of the CAP gene (or N region ofthe protein) being derived from AAV-3B. AAV1 and AAV6 are very similarto each other on amino acid level, and thus (as illustrated in FIG. 8),it is possible to look at AAV-PAEC as if it was derived from AAV-3B andAAV1, or from AAV-3B and AAV6; in each case, three amino acids differ inAAV-PAEC as compared to AAV1 or AAV6.

FIG. 9 shows a predicted 3D structure of AAV-PAEC (based on solvedstructure of part of AAV2 VP3 protein), assuming that AAV-PAEC iscomposed of sequences from AAV3B and AAV6 (see FIG. 8). The 3 mutationsat positions 418, 531 and 584 are shaded gray.

Example 2 In Vivo Studies

The AAV library generated according to the example above and screened toselect new AAV isolates in vitro in human pulmonary artery endothelialcells (hPAEC) was then screened in vivo in humanized FRG mice, accordingto methods described below.

Experimental Design of In Vivo AAV Library Selection

Similar to the in vitro AAV selection scheme described in Example 1, theAAV shuffle library was screened in FRG mice. As shown in FIG. 10,animals were injected with different amounts of AAV library, followed byinjection of a fixed amount of wtAd5 virus. After three days, animalswere sacrificed, and their livers extracted, homogenized and frozen inaliquots. One aliquot from each animal was used for analysis by westernblot using anti-VP1-2-3 CAP antibody for detection. Once the animal withthe lowest, but detectable signal, was identified, another frozenaliquot of liver from that animal was processed, and cleared liverlysate was injected in different amounts into another group of animals.As in the in vitro experiment described in Example 1, in order toinactivate hAd5 present in the lysate, the liver lysate was incubated at65 C for 30 min. prior to injection into another cohort of animals.

The experimental design shown in FIG. 10 was modified so that at eachround of selection one or two humanized FRG animals were used (FIG. 11).Liver lysates from each animal in the in vivo AAV selection wereanalyzed by CAP-specific PCR. As can be seen in FIG. 12, the CAPspecific band (˜2.2 kb) was present in all humanized FRG animals used inthe study, and the intensity of the signal is strong in all samples. Incontrol non-humanized FRG animals, the CAP specific signal is lost after3 rounds of selection, indicating that no AAV specific amplification wasobserved in those animals (this was expected, as human Ad5 virusrequired for AAV replication does not infect/replicate in mouse cells,but only in human cells, thus supporting AAV replication only in humanhepatocytes present in humanized FRG animals). This control furthershows that in humanized FRG animals the AAV selection took place inhuman hepatocytes but not in mouse cells.

With each round of selection, AAV DNA was isolated from liver lysate andused to sequence the CAP genes present in the pool. FIG. 13 comparesdata for (1) the AAV library injected into the first animal, (2) thelysate obtained from the first animal, and (3) the lysate from thefourth animal. At each round 100-150 clones were sequenced. Most of theclones sequenced from the library and from Mouse-1 were different fromeach other, showing high variability of the library at the early stagesof in vivo selection (such a large number of different clones wereidentified in the library and in Mouse-1 that they are represented as asolid black bar in the graphic representation shown here). In Mouse-4,however, over 24% of sequenced clones were of identical sequence,showing a positive selection for certain AAV clones present in the AAVlibrary.

Upon in vivo selection of rAAV serotypes on human hepatocytes in a mousewith a humanized liver, several novel rAAVs with high efficiency wereidentified and sequenced. Also valuable are specific novel rAAVserotypes selected in vitro in human pulmonary arterial endothelialcells (such as, for example, but not limited to AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12 and AAV-PAEC13isolates).

FIG. 14 illustrates a reconstruction of the genealogical relationshipbetween one of the in vivo isolates, AAV-LK01 and wildtype AAVs used togenerate the library (as shown in FIG. 1). AAV-LK01 is most closelyrelated to AAV8. The bottom of FIG. 14 graphically represents thepredicted origin of various regions of the new isolate based on theidentity of certain amino acid residues in these regions that correspondto residues within wildtype AAVs.

FIG. 15 shows a predicted 3-D structure of AAV-LK01 (based on solvedstructure of part of AAV2 VP3 protein). Point mutations indicated inFIG. 14 are shaded gray in the 3D model.

In order to better predict the characteristics of new in vivo-selectedAAV isolates and to gain some insight into the receptor used by suchisolates to bind/enter cells, we compared their sequences to wildtypeAAV-2, specifically looking at the amino acids responsible for heparinbinding. The residues implicated in heparin binding in AAV2 werecompared to residues in new isolates AAV-PAEC and AAV-LK01. The AAVsources of the amino acids in new AAVs at indicated positions are givenin parenthesis in FIG. 16. The whole region of the AAV-LK01 where thoseresidues are located was derived from AAV8, and thus one can predictthat AAV-LK01 will closely resemble AAV8 when it comes to the choice ofa receptor. AAV-PAEC, on the other hand, differed significantly fromAAV2, and each of the residues could have been derived from number ofother AAVs, with AAV1 and AAV6 contributing to each of the 6 residuesimplicated in heparin binding. Thus, AAV-PAEC might be predicted to usethe same or similar receptor as AAV1 and/or AAV6.

FIG. 17 shows a reconstruction of the genealogical relationship betweenthe top three in vivo-selected isolates, AAV-LK01, AAV-LK02 and AAV-LK03and wildtype AAVs used to generate the library. AAV-LK01 was found to bemost closely related to AAV8, AAV-LK02 to AAV1 and AAV6, and AAV-LK03 toAAV3B.

FIG. 18 graphically represents the sequence of AAV-LK02 and the identityof corresponding amino acid residues in the new isolate as compared tovarious wildtype AAVs. AAV-LK02 appears to be mostly derived from AAV1(or AAV6—due to very high % homology between AAV and AAV6), with theexception of a hyper-variable 50 amino acid (aa) region between aminoacid nos. 450-500. The table shows each of the mutations in the 50 aaregion in the left column, and indicates the possible parental wildtypeAAV from which this residue was derived in the right column. Severalmutations cannot be traced back to any of the parental AAVs, and weremost probably caused by random mutations during the PCR used to generatethe library, or were introduced during the viral replication, and arethus results of a natural viral evolution process.

FIG. 19 shows a predicted 3-D structure of AAV-LK02 (based on solvedstructure of part of AAV2 VP3 protein). Point mutations identified inFIG. 18 are shaded gray in this 3-D model.

FIG. 20 graphically represents of the sequence of AAV-LK03 and theidentity of corresponding amino acid residues in the new isolate ascompared to various wildtype AAVs. AAV-LK03 appears to be mostly derivedfrom AAV3B with the exception of a hyper-variable region in the 5″ ofthe gene and a single point mutation at the 3′ end of the gene. Thetable shows each of the mutations in the left column, and indicates thepossible parental wildtype AAV from which this residue was derived.Several mutations cannot be traced back to any of the parental AAVs, andwere most probably caused by random mutations during the PCR used togenerate the library, or were introduced during the viral replication,and are thus results of the natural viral evolution process.

FIG. 21 presents a reconstruction of the genealogical relationship (onthe DNA level) between the AAVs identified during in vivo libraryselection, and wildtype AAVs used to generate the library.

FIG. 22 presents a reconstruction of the genealogical relationship (onthe amino acid level) between the AAVs identified during in vivo libraryselection, and wildtype AAVs used to generate the library.

FIGS. 23A and 23B show a comparison of the new isolates (from selectionin PAEC cells as well as from in vivo selection), which were vectorized(i.e., packaged into non-infectious, non-replicating recombinantvectors) and further studied in a side-by-side comparison. Each AAVisolate and the ten wildtype AAVs used to generate libraries wereproduced in three independent small-scale productions (n=3) and thetiter of each vector was determined by extracting DNA from the vectorand quantifying it by dot blot using a known amount of DNA as astandard. Dot blot titers were determined for the isolates as well asthe wildtype AAVs. FIG. 23B presents this data in graphical form.

As shown in FIG. 24A, the vectorized AAV isolates and wildtype AAVs madein the three independent small-scale productions were used to transduceHuh7.5 cells, and the transduction titers determined. The vectorsexpressed eGFP under an hEF1 promoter. Transduction titer was calculatedusing the following formula: (% GFP/100)*dilution factor*cell number.FIG. 24B presents a graphic representation of the transduction titers onHuh7.5 cells. From the data, it can be seen that AAV-DJ is significantlybetter at transducing Huh7.5 cells than any of the wildtype AAVs. Of thenew isolates, AAV-LK03 was found to transduce Huh7.5 cells at least 10times better than AAV-DJ, and almost two logs better than any of thewildtype AAVs. AAV-PAEC is also significantly better at transducingHuh7.5 cells than any of the wildtype AAVs.

Dot blot titer is a physical titer that does not depend on the celltype, but rather represents the number of intact AAV particlescontaining AAV genome in the vector preparation. Using dot blot titerand the number of μl of each preparation used to transduce the cells,the transduction efficiency per number of viral genomes used fortransduction (Transduction titer/vg) can be calculated. In order tobetter compare the transduction efficiency of the new AAV isolates onHuh7.5 cells, the transduction efficiency was normalized to the vectortiters obtained from a dot blot. Results are presented in FIG. 25A. Theresults can also be represented by normalizing one of the vectors to avalue of 1 (assigning value of 1 to the vector with the lowesttransduction/vg). Results represented in this way are shown in FIG. 25B.FIG. 25B is a graphical representation of the transduction/vg on Huh7.5cells normalized to the vector with the lowest transduction/vg (in thiscase AAV-LK04). Presented thusly, the graph in FIG. 25B shows foldincrease in transduction/vg compared to AAV-LK04 which was assigned thevalue of 1. FIG. 25C is a graphical representation of thetransduction/vg for AAV isolates and wildtype AAVs, without normalizingto the weakest isolate. From these data, it can be seen that AAV-LK03 is30-times better at transducing Huh7.5 cells than AAV-DJ.

FIG. 26 is a graphical representation of the transduction/vg for variousAAV isolates and wildtype AAVs on 293 cells (data shown withoutnormalizing to the weakest isolate). These data show that AAV-LK06 is30-times better at transducing Huh7.5 cells than is AAV-DJ.

FIG. 27 is a graphical representation of the transduction/vg for AAVisolates and wildtype AAVs on NIH3T3 cells (data shown withoutnormalizing to the weakest isolate). These data clearly show that AAV-DJand AAV-PAEC are the most efficient vectors at transducing NIH3T3 cells.

FIG. 28 is a graphical representation of the transduction/vg forselected AAV isolates and wildtype AAVs on Mouse Embryonic Fibroblasts(MEF) cells (data shown without normalizing to the weakest isolate).These data clearly shows that AAV-DJ is the most efficient vector attransducing MEF cells.

The results of another dot blot titer of several rAAV isolates incomparison to wildtype AAVs are presented in FIG. 29.

The neutralizing effects of hepatocyte growth factor on some AAVs andrAAV isolates is illustrated in FIG. 30.

FIG. 31 shows the transduction efficiency of primary human hepatocytesby selected wildtype AAVs and rAAV isolates.

FIG. 32 compares the expression levels of recombinant human factor IX(FIX) in immunocompetent C57/BL6 mice injected with variousFIX-expressing AAVs.

FIG. 33 compares the ability of selected rAAV isolates and wildtype AAVsto avoid neutralization by human immune globulin (IVIG).

FIG. 34 compares transduction efficiency of certain rAAV isolates towildtype AAVs in 293 cells.

FIG. 35 compares transduction efficiency of certain rAAV isolates towildtype AAVs in Huh 7.5 cells.

FIG. 36 compares transduction efficiency of certain rAAV isolates towildtype AAVs in PAEC cells

FIG. 37 compares transduction efficiency of certain rAAV isolates towildtype AAVs in primary human keratinocytes.

FIG. 38 compares transduction efficiency of certain rAAV isolates towildtype AAVs in DND-41 cells.

FIG. 39 compares transduction efficiency of particular rAAV isolates towildtype AAVs in DND-41 cells.

FIG. 40 compares transduction efficiency of certain rAAV isolates towildtype AAVs in 3T3 cells.

FIG. 41 compares transduction efficiency of certain rAAV isolates towildtype AAVs in HeLa cells.

FIG. 42 compares transduction efficiency of certain rAAV isolates towildtype AAVs in MEF cells.

FIG. 43 compares transduction efficiency of certain rAAV isolates towildtype AAVs in H4TG cells.

FIG. 44 compares transduction efficiency of certain rAAV isolates towildtype AAVs in LMH cells.

FIG. 45 compares transduction efficiency of certain rAAV isolates towildtype AAVs in FRhK-4 cells.

FIG. 46 compares the transduction of various cell lines by several wildtype and recombinant AAV vectors carrying an eGFP expression cassette.Transduction titer [TU/ml] was normalized to the dot blot titer [vg/ml].For each cell line, the vector with the lowest normalized transduction[TU/vg] was assigned the value 1 and used as a basis for normalizationof all other vectors. The vector with the highest normalizedtransduction level for each cell line are shown in bold. Nineteen humanhepatocyte-selected clones were tested.

Several rAAVs were identified by selection on human PAEC cells; thesewere designated as AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7,AAV-PAEC8, AAV-PAEC11, AAV-PAEC12 and AAV-PAEC13. FIG. 47 presents aschematic of the genealogical relationship on the DNA level and aminoacid level between certain of these AAV-PAEC recombinants and thewildtype AAVs used to generate the library.

FIG. 48 shows the titer and transduction efficiency of several rAAVisolates obtained by selection on human PAEC cells as compared towildtype AAVs.

In summary, several of the rAAVs were found to have increasedtransduction efficiency of many cell types, including primary humanhepatocytes and primary human keratinocytes, compared to the previouslyknown and efficient AAV-DJ. In some embodiments, the rAAVs were found tohave at least 10 to 50 times greater transduction efficiency thanAAV-DJ. Also, rAAV vectors were found to have improved resistance toneutralization by pooled human immune globulin relative to AAV-2(currently used in clinical settings) and AAV-DJ. Such resistance isreasonable, given that the rAAV capsid was selected from a librarypartially based on its ability to produce virus that resistsneutralization by human immune globulin. The AAV capsid selectionmethods and novel rAAVs identified herein and using these methods solvethe problem of lack of efficacy exhibited by many gene transfer and genetherapy approaches.

While a number of exemplary aspects and embodiments have beenillustrated and described, those of skill in the art will recognizecertain modifications, permutations, additions and sub-combinationsthereof that can be made without departing from the spirit and scope ofthe invention(s). It is therefore intended that the following appendedclaims and claims hereafter introduced are interpreted to include allsuch modifications, permutations, additions and sub-combinations as arewithin their true spirit and scope, and it is to be understood that thedisclosure is not to be limited to the specific embodiments disclosedherein, as such are presented by way of example. Although specific termsare employed herein, they are used in a generic and descriptive senseonly and not for purposes of limitation.

All literature and similar materials cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, internet web pages and other publications cited in thepresent disclosure, regardless of the format of such literature andsimilar materials, are expressly incorporated by reference in theirentirety for any purpose to the same extent as if each were individuallyindicated to be incorporated by reference. In the event that one or moreof the incorporated literature and similar materials differs from orcontradicts the present disclosure, including, but not limited todefined terms, term usage, described techniques, or the like, thepresent disclosure controls.

What is claimed is:
 1. A recombinant viral vector, comprising: a capsidprotein with an amino acid sequence, relative to wildtype AAV3B, thathas (i) at least 3 amino acid substitutions in the N-terminal regioncomprised of the about the first 105 amino acid residues and (ii) and atleast 1 amino acid substitution in a region of the protein correspondingto the amino acid residues in the protein from residue 106 to the Cterminus, wherein the vector transduces cells in vitro at least 10 timesbetter than AAV-DJ.
 2. The recombinant viral vector of claim 1, whereinthe capsid protein has an amino acid sequence, relative to wildtypeAAV3B, that has at least 4 amino acid substitutions in the N-terminalregion comprised of the about the first 105 amino acid residues.
 3. Therecombinant viral vector of claim 1, wherein the capsid protein has anamino acid sequence, relative to wildtype AAV3B, that has at least 5amino acid substitutions in the N-terminal region comprised of the aboutthe first 105 amino acid residues.
 4. The recombinant viral vector ofclaim 1, wherein the capsid protein has an amino acid sequence, relativeto wildtype AAV3B, that has at least 6 amino acid substitutions in theN-terminal region comprised of the about the first 105 amino acidresidues.
 5. The recombinant viral vector of claim 1, wherein the atleast 3 amino acid substitutions in the N-terminal region comprises aglutamine substitution relative to wildtype AAV3B at position
 26. 6. Therecombinant viral vector of claim 1, wherein the at least 3 amino acidsubstitutions in the N-terminal region comprises an alanine substitutionrelative to wildtype AAV3B at position
 29. 7. The recombinant viralvector of claim 1, wherein the at least 3 amino acid substitutions inthe N-terminal region comprises a lysine substitution relative towildtype AAV3B at position
 31. 8. The recombinant viral vector of claim1, wherein the at least 3 amino acid substitutions in the N-terminalregion comprises an alanine substitution relative to wildtype AAV3B atposition
 42. 9. The recombinant viral vector of claim 1, wherein the atleast 3 amino acid substitutions in the N-terminal region comprises analanine substitution relative to wildtype AAV3B at position
 67. 10. Therecombinant viral vector of claim 1, wherein the at least 3 amino acidsubstitutions in the N-terminal region comprises a lysine substitutionrelative to wildtype AAV3B at position
 105. 11. The recombinant viralvector of claim 1, wherein the at least 1 amino acid substitution in theprotein from residue 106 to the C terminus comprises a prolinesubstitution relative to wildtype AAV3B at position
 735. 12. Arecombinant viral vector, comprising: a capsid protein with an aminoacid sequence comprising, relative to wildtype AAV3B, one or more of asubstitution at amino acid residues 26, 29, 31, 42, 67, 105 and/or 735,wherein the vector transduces cells in vitro at least 10 times betterthan AAV-DJ.
 13. The recombinant viral vector of claim 12, wherein theamino acid sequence comprises a glutamine substitution relative towildtype AAV3B at residue
 26. 14. The recombinant viral vector of claim12, wherein the amino acid sequence comprises an alanine substitutionrelative to wildtype AAV3B at residue
 29. 15. The recombinant viralvector of claim 12, wherein the amino acid sequence comprises a lysinesubstitution relative to wildtype AAV3B at residue
 31. 16. Therecombinant viral vector of claim 12, wherein the amino acid sequencecomprises an alanine substitution relative to wildtype AAV3B at residue42.
 17. The recombinant viral vector of claim 12, wherein the amino acidsequence comprises an alanine substitution relative to wildtype AAV3B atresidue
 67. 18. The recombinant viral vector of claim 12, wherein theamino acid sequence comprises a lysine substitution relative to wildtypeAAV3B at residue
 105. 19. The recombinant viral vector of claim 12,wherein the amino acid sequence comprises a proline substitutionrelative to wildtype AAV3B at residue
 735. 20. A method for transductionof a gene of interest, comprising, providing a recombinant viral vectorhaving a capsid protein that transduces cells in vitro 10-fold higherthan AAV-DJ, where the capsid protein comprises mutations at positions26, 29, 31, 42, 67, 105 and 735, relative to wildtype AAV3B.